The Extracellular Adherence Protein from Staphylococcus aureus Inhibits the Classical and Lectin Pathways of Complement by Blocking Formation of the C3 Proconvertase

Abstract

The pathogenic bacterium Staphylococcus aureus actively evades many aspects of human innate immunity by expressing a series of small inhibitory proteins. A number of these proteins inhibit the complement system, which labels bacteria for phagocytosis and generates inflammatory chemoattractants. While the majority of staphylococcal complement inhibitors act on the alternative pathway (AP) to block the amplification loop, only a few proteins act on the initial recognition cascades that constitute the classical (CP) and lectin (LP) pathways. We screened a collection of recombinant, secreted staphylococcal proteins to determine if S. aureus produces other molecules that inhibit either the CP and/or LP. Using this approach, we identified the extracellular adherence protein (Eap) as a potent, specific inhibitor of both the CP and LP. We found that Eap blocked CP/LP-dependent activation of C3, but not C4, and that Eap likewise inhibited deposition of C3b on the surface of S. aureus cells. In turn, this significantly diminished the extent of S. aureus opsonophagocytosis and killing by neutrophils. This combination of functional properties suggested that Eap acts specifically at the level of the CP/LP C3 convertase (C4b2a). Indeed, we demonstrated a direct, nanomolar-affinity interaction of Eap with C4b. Eap binding to C4b inhibited binding of both full-length C2 and its C2b fragment, which indicated that Eap disrupts formation of the CP/LP C3 pro-convertase (C4b2). As a whole, our results demonstrate that S. aureus inhibits the two initiation routes of complement by expression of the Eap protein, and thereby define a novel mechanism of immune evasion.

The Group B Streptococcus–Secreted Protein CIP Interacts with C4, Preventing C3b Deposition via the Lectin and Classical Complement Pathways

“…Woehl et al (17) observed that a knockout mutant carrying an in-frame deletion of Eap did not show reduced levels of bacterial C3b deposition or phagocytosis compared with wild-type, whereas a protective effect was achieved when the protein was added exogenously, as we observed for GBS CIP. The data suggested that exogenous Eap, but not surface-retained Eap, could significantly contribute to S. aureus complement evasion.…”

“…The mechanism by which CIP interferes with CP and LP activation is reminiscent of the recently described effect of the Eapsecreted protein expressed by S. aureus. Indeed, Woehl et al (17) demonstrated a direct nanomolar affinity interaction of Eap FIGURE 9. CIP interferes with GBS killing in a whole-blood assay.…”

“…Studies in the last decade have revealed that another Grampositive pathogen, Staphylococcus aureus, produces soluble proteins interfering with the activation of the complement system such as staphylococcal complement inhibitor (14), extracellular fibrinogenbinding protein (Efb) (15,16), and extracellular adherence protein (Eap) (17). We hypothesized that S. agalactiae might secrete similar yet unidentified complement regulators assisting the bacteria to escape phagocytic killing.…”

Evasion and interactions of the humoral innate immune response in pathogen invasion, autoimmune disease, and cancer

“…Proteins expressed on the S. aureus bacterial surface prevent formation of the MAC and bacterial lysis [7, 9]. In addition, at least four S. aureus proteins bind to C3b and prevent the formation of further convertases [8, 14, 16], while an additional S. aureus protein prevents formation of the C3 convertase halting the complement cascade [18]. …”

Identification of peptidic inhibitors of the alternative complement pathway based on Staphylococcus aureus SCIN proteins

“…(Garcia et al, 2012a; Kang et al, 2013; Woehl et al, 2014)]. Among these factors are the staphylococcal complement inhibitors SCIN-A and SCIN-B/C (Jongerius et al, 2007; Rooijakkers et al, 2005).…”